L12: Coagulation in the lab and bleeding disorders

Question 1

APPT

Answer 1

= activated partial thromboplastin time

• Measures intrinsic pathway (contact factors, not physiological)
• All factors except VII
• Mostly used to assess XII (prolonged APTT without bleed), XI, IX and VIII
• Normal range 34-37 seconda
• Commonly prolonged in haemophilias due to reduced VIII and IX

Question 2

APTT method

Answer 2

1. Venous sample collected into citrate (citrate removes calcium to prevent clotting)
2. Spin sample down to collect plasma
3. Add phospholipid and an activator
4. Add calcium to overcome citrate
5. Measure length of time to clot formation (look at light absorbance)

Requires: contact factor (kaolin, silica, ellagic acid), source of phospholipid, calcium

Question 3

APTT mixing studies

Answer 3

ATPP, PR, TCT basic screening tests
If APTT prolonged –> additional mixing studies done

• 1:1 mix with normal plasma and incubation
• Provides normal clotting factors
• If APTT corrected and remains normal = factor deficiency
• If APTT does not fully correct = inhibitor present

Question 4

PR method

Answer 4

= prothrombin ratio

• Add tissue factor to stimulate extrinsic pathway
• Generates thrombin by Xa converting II to IIa (bypasses complex 2: VIII and IX, does not require amplification phase)
• Prolonged in people with deficiency of VII, X, V, prothrombin or fibrinogen
• Normal prothrombin time 12-15secs

Question 5

PR ratio

Answer 5

• Standardises test to account for different lab methods and different normal ranges for PT
• PT (patient)/PT (normal plasma)
• Normal ratio ~1.0 (0.8-1.2)
• Ratio used to monitor therapy with oral warfarin (corrected PR for warfarin monitoring = INR)

Question 6

TCT

Answer 6

= Thrombin clotting time

• Thrombin added to plasma: fibrinogen -> fibrin
• Standard TCT used to measure fibrinogen (normal 4-10 secs)
• Dilute TCT used to measure thrombin inhibitors: heparin and dabigatran (normal 15-20 secs)

Question 7

Natural inhbitors

Answer 7

Antithrombin, protein C and protein S shut down coagulation but DO NOT affect APTT

Question 8

Causes of prolonged APTT 1+1

Answer 8

1. Lupus anticoagulant
2. Heparin
3. Dabigatran
4. Factor inhibitors (rare)

Question 9

Lupus anticoagulant

Answer 9

• Plasma antibodies interfere with phospholipid and prolong APTT and 1+1 but does not cause bleeding
• Can present transiently in patients who are unwell (infection or inflammation)
• OR may be part of antiphospholipid syndrome (disorder with excess clotting, sometimes recurrent miscarriage)

Question 10

Factor inhibitors

Answer 10

• Autoimmune antibodies against clotting factor (usually VII)
• Associated with bleeding including bruising
• Can be life-threatening
• Rare but should be considered in bleeding

Question 11

Heparin

Answer 11

• Confirm by addition of protamine (reverses effect of heparin)
• Anticoagulant that works as inhibitor by upregulating anti-thrombin
• Biological glycosaminoglycan chains
• Natural GAGs activate anti-thrombin in vivo but do not affect APTT, heparin infusion is a much larger amount
• Commonly used to lock central lines, potential for contamination if tube collected in this way
• TCT very prolonged (corrects with protamine)

Question 12

Dabigatran

Answer 12

• Direct inhibitor of thrombin
• Oral tablets, usually for atrial fibrillation
• Does not correct with protamine
• TCT very prolonged

Question 13

APTT prolonged, PT normal

Answer 13

Deficiencies of factor(s) VIII, IX, XI, XII (intrinsic)

• XI mild bleeding disorder
• XII asymptomatic
• More serious bleeding likely VIII or IX

Question 14

PT prolonged, APTT normal

Answer 14

• Deficiency of factor VII (extrinsic)

- Occasionally mild deficiency of II, V, X, fibrinogen (PT mildly prolonged)

Question 15

Both PT and APTT prolonged

Answer 15

• Deficiency of factor(s) II, V, X and I (common)

- Multiple factor deficiencies

Question 16

TCT prolonged

Answer 16

• Deficiency of fibrinogen

- Thrombin inhibitor

Question 17

Other tests that can be done

Answer 17

• Single factor assays (measure factor VIII and IX)
• Whole blood clotting tests
• Primary haemostasis (platelet function testing)
• D-dimers

Question 18

Multiple factor deficiencies

Answer 18

Most common scenario

• Warfarin or vit K deficiency (II, VII, IX, X)
• Massive blood loss (loss of coagulation factors and dilution with fluid)
• DIC: widespread activation of coagulation causing thrombosis then bleeding, low fibrinogen often seen (e.g. meningicoccus)
• Liver disease: lack of production of coagulation factors and inhibitors (except VIII which is also produced by endothelial cells lining blood vessels)

Question 19

Prolonged PT

Answer 19

• Extrinsic pathway
• Warfarin (low II, VII, IX, X)
• Vit K deficiency
• Liver disease
• Low factor VII (APTT normal)

Question 20

Warfarin

Answer 20

• Inhibits vit K recycling (lack of carboxylated factors) so reduces II, VII, IX, X
• Dose adjustments via INR
• Reversed by vit K in 12hrs (or more rapidly by clotting factor replacement using plasma products)
• APTT prolonged, not routinely measured
• Used in AF, venous thromboembolism and other thrombotic disorders

Question 21

Hereditary factor deficiencies

Answer 21

• RARE
• Usually single factor is deficient
• First: have a bleeding history
• Basic coagulation tests and depending on these do single factor assays

Question 22

Haemophilia clinical presentation

Answer 22

Severe disease
Spontaneous joint bleeds:
- Chronic arthropathy
- Joint destruction
- Deformity 
- Arthritis

Soft tissue bleeds:

• Tissue damage
• Nerve damage
• Deformity

Question 23

Haemophilia A

Answer 23

• Factor VIII deficiency
• Most common hereditary disease with severe bleeding
• 1/5000 live male births (females can have mild)
• X-linked recessive

Question 24

Haemophilia B

Answer 24

• Deficiency of factor IX
• X-linked recessive
• Rare, 1/30,000 live male births
• Clinical features identical to haemophilia A

Question 25

Treatment of haemophilia

Answer 25

• Replace missing factor
• Recombinant in NZ (factor VIII and IX)
• Prophylaxis in children and teenagers when severe
• Normal lives and joint outcomes (except patients who develop inhibitors)

Question 26

Testing for haemophilia

Answer 26

• Prolonged APTT with normal PR
• Single factor assay (low VIII or IX)
• Genetic analysis (sequencing of factor VIII or IX gene)

Question 27

Von Willebrand factor

Answer 27

• Plasma glycoprotein
• Synthesised in megakaryocytes and endothelial cells
• Promotes platelet adhesion at vessel wall
• Carrier for VIII and stabilises preventing degradation

Question 28

Von Willebrands disease

Answer 28

• Most common inherited bleeding disorder (1-3%)

- Autosomal dominant

Question 29

Test for VW disease

Answer 29

• Abnormal platelet function screen
• Marginally prolonged APTT
• Low factor VIII (normal VIII does not exclude VW disease)

Question 30

Clinical presentation of VW disease

Answer 30

• Mucosal bleeding (epistaxis, gum bleeds, GI bleeds)
• Menorrhagia
• Postpartum or peri-operative bleeding
• Autosomal family history
• Can be mild to very severe

Question 31

DIC

Answer 31

= disseminated intravascular coagulation

• Acquired syndrome by intravascular activation of coagulation, loss of clot localisation
• Red cell fragments in blood film (sheared apart due to obstruction of vessels by clot)
• Caused by sepsis, malignancy, organ damage (e.g. pancreatitis, trauma